Process for extracting minor fatty compounds from a substance of biological origin

ABSTRACT

A method for extracting minor fatty compounds, particularly steroids, from biological matter by means of cyclodextrin. According to the method, the cyclodextrin is stirred into said biological matter in a water-containing reaction medium to allow the formation of inclusion complexes between the cyclodextrin and the minor fatty compounds, whereafter said complexes are separated from said matter, the amount of water present in the reaction medium being lower than 100 % wt. in relation to the matter to be treated. The temperature of the reaction medium and the amount of water present therein are such that the cyclodextrin is partially undissolved.

TECHNICAL FIELD

The present invention relates to a process for extracting minor fattycompounds, especially steroids, and more particularly cholesterol, whichoccur in a substance of biological origin.

BACKGROUND OF THE INVENTION

"Minor fatty compounds" designates all non-glyceride fatty constituentswhich occur in natural fatty substances. They may be steroids,especially sterols, fatty alcohols, fat-soluble vitamins, pigments,hydrocarbons, free fatty acids, abietic and pimaric acids, flavouringcompounds and the like.

For more detail, reference can be made to the definition given in"Manuel d'analyse des corps gras (Manual of the analysis of fattyacids)" JP WOLF--Ed Azoulay--Paris 1969, pages 11 and 25.

Within the context of the present invention, the term "steroids"designates all compounds containing a more or less hydrogenatedcyclopentanophenanthrene skeleton. Many of these compounds are alcoholsand are termed sterols. They may occur in the form of esters especiallyof fatty acids, commonly designated by the term "cerides".

Cholesterol belongs to the sterol family. It has been known for sometime that it is the main constituent of biliary calculi. Its reputationhas recently been strengthened by its involvement in circulatorydisorders, and more particularly the hardening of the arteries.

Nonesterified cholesterol is the main constituent of fatty substances ofanimal origin. These animal fats, which are present in most of ourfoodstuffs, constitute an important source of cholesterol which, in theevent of an excess, may be the cause of serious cardiovascular diseasesincluding myocardial infarction in particular.

Fatty substances of plant origin contain not cholesterol butphytosterols whose structure is very similar to it, such as for example:β-sitosterol, campesterol, stigmasterol, brassicasterol,Δ-7-stigmasterol, Δ-5-campesterol, Δ-5-avenasterol, Δ-7-avenasterol,Δ-7-9-sigmatadienol, fucosterol or ergosterol.

The process according to the invention also applies to the oxidisedderivatives of steroids: sterones. These compounds occur especially infrying oils used repeatedly. Their presence in food products is notdesirable as they are not devoid of toxic effect.

In addition, steroids are products of high added-value both economicallyand technically. Indeed, they have the potential of constituting highgrade raw materials for the preparation of vitamin compounds(ergosterol: vitamin D2) or hormonal compounds (equilenin, oestrone,progesterone, testosterone or cortisone and the like). In addition totheir pharmaceutical applications, steroids, and more particularlysterols and their fatty acid esters (cerides), may be used in thecosmetics industry as emulsifiers.

As far as the other minor fatty compounds are concerned, there may bedistinguished, on the one hand, compounds which are undesirable from thefood or technological point of view such as free fatty acids whichpromote oxidation, degradation compounds such as polymerised acids,foul-smelling compounds, colouring substances and savoury substances andthe like; and, on the other hand, compounds of high food ortechnological value such as fat-soluble vitamins, essential fatty acids(linoleic acid, α-linoleic acid, γ-linoleic acid, arachidonic acid, DHAand EPA), rare fatty acids (ricinoleic and abietic acids), andflavouring compounds.

There are thus several Justifications for all the technologicaldevelopments designed to extract the minor fatty compounds from fattybiological substances. This term designates in particular, on the onehand, animal fats, what they are storage fats such as fish oils oranimal oils (tallow, chicken fat, lard and the like), structural fatssuch as those present in muscular, placenteral or nerve tissue (meat,egg, spinal cord, brain and the like) or fats from glandular secretionsuch as milk fats, or fats from skin secretion such as cerides(lanolin); and, on the other hand, vegetable fats. Finally, the fats maybe a mixture of animal fats and vegetable fats such as margarines.

DESCRIPTION OF THE RELATED ART

Various methods for extracting minor fatty compounds from fat-containingbiological substances have thus already been proposed.

Primarily, techniques exist for extracting steroids, especiallycholesterol.

One of them consists in placing fatty substance of animal origin incontact with digitonin (digitogenin glucodise), which has the propertyof reacting with cholesterol to give an insoluble precipitate. Theefficiency and the results of this method are not satisfactory due tothe difficulty of separating the precipitate from the mixture. And inany case, this method is inapplicable industrially, especially for foodproducts.

Cholesterol may also be extracted from fatty substances by entrainmentby means of a solvent. The main disadvantage of this process is that thesolvents generally used are toxic and that traces of them always remainin the fatty substances considered.

Processes of microdistillation, which are inapplicable at the industriallevel, or alternatively of absorption on columns such as those describedfor example in European Patent Applications No. 0,174,848 and No.0,318,326, are also known. These applications describe a processaccording to which the fatty substance, maintained in a liquid state,passes through an absorption column, in this case activated charcoal. Itis evident that such a process is very hard to implement and,furthermore, the extraction which is achieved by it is not veryselective.

Another physicochemical process for extracting cholesterol from fats isdisclosed by Japanese Patent Application No. 59/140,299. It consists inplacing a substance charged with cholesterol, such as milk powder, incontact with supercritical CO₂ at a temperature of between 35° and 45°C. and at a pressure of between 130 and 200 atm. Obtaining thesephysical conditions requires the use of complex and expensive equipment.Control of the process is thus very intricate as a result. Furthermore,as specified in the patent application, other lipid compounds areentrained by the supercritical CO₂. This process is not thereforeselective.

A process for the biodegradation of the said sterols, which is describedby European Patent Application No. 0,278,794, has also been conceivedfor removing sterols from fats. This process uses bacteria which, whenplaced in contact with the fatty substance, are capable of metabolisingthe sterols contained therein. Like all processes involvingfermentations, this biodegradation process is very intricate to managebecause of the inherent variability of living matter. Furthermore, theequipment used and the relatively long duration are, inter alia,elements which make such a process expensive. Finally, the catabolitesproduced during these fermentations remain up until now completelyunknown with respect to their nature and their toxicity and are, in anycase, present in the fatty substance thus treated.

A process for removing the cholesterol present in a fatty substance ofanimal origin is also disclosed by European Patent Application No.0,256,911. It is based on the property, known since 1958, which isexhibited by cyclodextrins (cyclic polyglucose compounds of tubularfrustoconical conformation containing 6, 7 or 8 glucose units andrespectively designated by alpha-, beta- or gamma-cyclodextrin) wherebythey accept sterol molecules, and especially cholesterol molecules, intotheir hydrophobic cavity to form inclusion complexes which are solublein water. According to this process, the fatty substance, which ismaintained fluid, is placed in contact, in an aqueous medium, with acyclodextrin, with stirring and under a nonoxidising atmosphere for 30minutes to 10 hours so as to allow the formation of complexes.Separation of the latter is then carried out by entrainment with waterwhich solubilises these complexes. The aqueous solution thus obtained isthen collected after decantation. The lipid phase obtained must bewashed with water several times so as to remove the traces of complexes.The conditions for carrying out this process turn out to be relativelyexacting. One of the main constraints to be met is the maintenance of anitrogen-based nonoxidising atmosphere over the entire period duringwhich the compounds are placed in contact. This period is moreover verylong in practice since, if reference is made to the examples, it can beobserved that it is always greater than or equal to 3 hours.

The yield of cholesterol extraction by this process is not very high.Indeed, in the best case, it is only 41%, and this after threesuccessive extractions as indicated in Example 3 of the description ofthis European patent application.

The amount of water required for the placing in contact beforeseparation corresponds to not less than 1000% by weight relative to thefatty substance to be treated.

Still on the same principle of using cyclodextrins for the extraction ofsteroids, European Patent Application No. 0,326,469 describes a processfor removing steroidal compounds present in a substance of biologicalorigin. According to this process, the substance of biological origin,which is made fluid if it is a solid fat at normal temperature, isplaced in contact with a cyclodextrin in aqueous medium. The contactingis carried out with stirring for 5 hours at a temperature of the orderof 40° C. A separation of the cyclodextrin/steroid complexes is thencarried out. Here again, it should be noted that the actual duration ofthe placing in contact, which is given in all the examples, is verylong: minimum of 5 hours.

The low yields of sterol extraction given in the examples are evidencethat this process lacks efficiency.

During the placing in contact before separation, the reaction mediumcontains more than 100% by weight of water relative to the substance tobe treated expressed on a dry basis.

Secondly, processes for extracting minor fatty compounds other thansteroids have been considered.

Among these processes, the one described in U.S. Pat. No. 3,491,132 anddesigned to reduce the free fatty acid content of glyceridic oils usingcyclodextrin may be mentioned. In this case, the cyclodextrin is placedin contact with the fatty medium in the presence of substantial amountsof water ranging from 200 to 1000% relative to the fatty medium treated.

These last three processes, which are described in European PatentApplications Nos. 0,256,911 and 0,326,469 and in U.S. Pat. No.3,491,132, have at least one major disadvantage in common, namely thatthey take place in aqueous medium and require very large amounts ofwater for the placing in contact.

It is evident that the use of such amounts of water constitutes ahandicap from the industrial point of view since it makes it necessaryto handle large reaction volumes and requires equipment of largecapacity which is therefore bulky and more expensive. Moreover, inaddition to the high direct cost which this entails, the treatment ofthe effluents obtained after the separation should also be taken intoaccount; this treatment representing a burden which increases as afunction of the amount of effluent.

In view of the foregoing, it should be noted that the techniques forextracting minor fatty compounds using cyclodextrin, which are the mostsatisfactory nevertheless, especially with respect to the quality of theproducts obtained, have not been optimised from the point of view ofeconomic and technical profitability.

SUMMARY OF THE INVENTION

Anxious to promote technical development in this field, the applicantcompany carried out long and numerous studies at the end of which theysucceeded, to their credit, in finding that it was preferable andadvantageous to carry out the placing in contact with small amounts ofwater, in other words, not to provide for the solubilisation of all thecyclodextrin used for placing in contact, by choosing a suitabletemperature and amount of water.

Accordingly, the present invention relates to a process for extractingminor fatty compounds, especially steroids, and more particularlycholesterol, which occur in a substance of biological origin, usingcyclodextrin, in which the cyclodextrin is placed in contact, withstirring and in a reaction medium containing water., with the substanceto be treated so as to allow the formation of inclusion complexesbetween the cyclodextrin and the minor fatty compounds, these complexesbeing subsequently separated from the said substance, characterised inthat the amount of water is less than 100% by weight relative to the drysubstance to be treated and in that, during the placing in contact, thetemperature of the reaction medium and the amount of water present inthe said medium are chosen so that the cyclodextrin is in a form whichis partially undissolved.

DETAILED DESCRIPTION OF THE INVENTION

By adjusting these two parameters, which amounts to providing acyclodextrin concentration in the reaction medium during the placing incontact which is above the maximum solubility of cyclodextrin at thereaction temperature, and by thus limiting the amounts of water used,all the costs of implementing the extraction process are substantiallyreduced while making the use more flexible and easier.

Furthermore, the extraction yields are improved for much shorterdurations of placing in contact, and the amounts of effluents collected,and therefore to be retreated, are substantially reduced. Theseeffluents are in concentrated form, which keeps down the costs oftransportation and retreatment.

The arrangements according to the invention are particularly novel asthey run counter to the teaching of prior extraction techniques usingcyclodextrins, according to which it was envisaged to use water insufficient amount to solubilise the bulk of the cyclodextrin. It wasthen assumed that the complexation could occur only if the cyclodextrinwas in soluble form.

And it is surprising and unexpected to have established, in conformitywith the present invention, that the complexation proceeds in an evenmore satisfactory manner from the point of view of the yields and theduration when the cyclodextrin is only partially solubilised, andtherefore essentially in suspension in the water.

This is contrary to the teaching of European Patent Application No.387,708, unpublished at the time of filing the present application,which relates to the extraction of cholesterol and fatty acids from ananimal fat using cyclodextrin and which envisages that the latter mustbe completely in dissolved form during the placing in contact.

In practice, knowing the reaction temperature for the placing incontact, the appropriate cyclodextrin concentration is determined bytaking care to choose it to be above the maximum solubility ofcyclodextrin at this temperature.

For this, reference is made to the solubility constants of cyclodextrinin water, as a function of the temperature. The table below, which istaken from "Cyclodextrins and their inclusion complexes" (page 33)Akademiai Kiado, Budapest 1982--J. SZEJTLI, gives the maximum solubilityvalues measured for beta-cyclodextrin.

    ______________________________________                                        Grams of β-cyclodextrin                                                                    Temperature in                                              in 100 ml of water                                                                              degrees Celsius                                             ______________________________________                                        1.20              15                                                          1.48              20                                                          1.79              25                                                          2.25              30                                                          3.51              40                                                          4.64              50                                                          6.05              55                                                          7.49              60                                                          10.18             65                                                          12.03             70                                                          14.80             75                                                          19.66             80                                                          ______________________________________                                    

For a given reaction temperature, a "saturation level" of cyclodextrinin solution in water is defined which corresponds to the followingratio: ##EQU1##

In conformity with one of the essential features of the presentinvention, the saturation level of the cyclodextrin present in thereaction medium, during the placing in contact, is greater than or equalto 1.67, preferably greater than or equal to 1.87, and still morepreferably greater than or equal to 2. A value less than 3000 isgenerally chosen, preferably less than 1500 and still more preferablyless than 750.

In the examples of European Patent Application No. 0,387,708, the amountof water used is always not less than 100% by weight relative to thesubstance to be treated expressed on a dry basis, and the saturationlevel is always less than 1.55.

The process according to the invention may be applied to natural fattysubstances which are essentially composed of triglycerides but low intotal phospholipids: that is to say having a total phospholipid contentof less than 10% by weight relative to the total lipids.

This process permits, in particular, the extraction of steroids such ascholesterol from numerous biological substances. They may be animal fatsand/or vegetable fats among which there may be mentioned:

milk fats such as concentrated or nonconcentrated butter;

animal fats such as tallow, lard, horse fat, chicken fats, fish oils,lanolin;

vegetable fats such as hydrogenated vegetable fats, palm oil, copra oil,cacao butter;

and mixtures thereof such as margarines.

In addition to removing these compounds from foodstuffs, the processconforming to the invention offers a useful source of steroids which arecapable of being used in the preparation of pharmaceutical ingredients,especially hormonal and vitamin ingredients, or in the cosmeticsindustry as emulsifiers.

The cyclodextrin used may be of the alpha, beta or gamma type,preferably of the beta type, which is substituted or unsubstituted. Themono- or poly-substituent groups of the cyclodextrin may be inparticular alkyl groups such as the hydroxypropyl or ethyl group, orglycoside or maltosyl type saccharides and the like. Cyclodextrinrendered insoluble in water, for example by polymerisation, may also bechosen.

Advantageously, the cyclodextrin used in the process according to theinvention is provided in pulverulent dried form. According to a specificembodiment, it has a water content of less than or equal to 11% byweight, preferably less than or equal to 7% by weight, and still morepreferably less than or equal to 5% by weight. It is optionallyrehydrated up to a water content of less than or equal to 15% by weight.

In conformity with an advantageous arrangement of the present invention,the amount of water present in the reaction medium, during the placingin contact and before separation, is preferably between 10 and 90%, andstill more preferably between 20 and 80%, by weight relative to thesubstance to be treated expressed on a dry basis.

It is worthwhile to note that the placing in contact takes place underan ambient atmosphere. Indeed, the process conditions are not harsh anddo not carry the risk of bringing about degradations, such as oxidation,of the sensitive fatty compounds (unsaturated fatty acids). Betterstill, cyclodextrin makes it possible, in addition, to remove, at leastpartially, the undesirable prooxidant compounds which may be present.

In conformity with a preferred embodiment of the invention, the placingin contact of cyclodextrin/substance to be treated is carried out at atemperature which is chosen such that the fatty substances present inthe said substance are in a"congealed-state.

Congealed state, within the context of the present invention, isunderstood to mean a state in which the level of solid or crystallinefatty substances is greater than or equal to 5% by weight.

This solid fat level is a common variable in the field of lipidtechnology. It is measured by low resolution Nuclear Magnetic Resonance,using pulsed minispectrometers.

Pulsed NMR, which is based on the fact that fat molecules in the liquidphase have a degree of mobility which is greater than that of themolecules of the solid phase, enables the percentage of solid or liquidfat to be determined. The principle and the applications of this methodare described for example in the journal R.I.B.C. of January-February,1985, No. 80, pages 23 to 26, or in the journal I-A-A of May, 1988,pages 463 to 471 or of June, 1988, pages 463 to 470.

Contrary to what is recommended in the prior art and especially inEuropean Patent Applications Nos. 0,256,911 and 0,326,469 and U.S. Pat.No. 3,491,132 which are mentioned above, the applicant company hasdemonstrated, surprisingly and unexpectedly, that the congealed state ofthe fatty substances which constitute the substance to be treatedcorresponds to an advantageous state for complexing-minor fattycompounds, especially steroids, with cyclodextrin at the stage ofplacing in contact with stirring.

Preferably, the operating conditions are chosen such that the level ofsolid fats is greater than or equal to 10%, and still more preferablygreater than or equal to 30%.

In this preferred embodiment, the substance to be treated, water - ifthe said substance to be treated does not contain it or does not containenough of it--and then cyclodextrin, preferably in pulverulent form, areplaced together in a mixer equipped with kneading means. The sequence inwhich the products are introduced is preferably the one indicated abovebut it is also possible, among all the possible variants, to mix thesubstance to be treated and water beforehand.

Advantageously, the mixture is provided in a pasty form which issubjected, during the entire period of placing in contact, to a kneadingoperation during which the formation of cyclodextrin/minor fattycompound complexes occurs.

For the separation of these complexes, and this is one of the noveltiesof this preferred embodiment of the process according to the invention,the kneaded paste is heated to a temperature which is chosen such thatthe fatty substances constituting the substance to be treated are in amolten state, that is to say in a state characterised by a level ofsolid or crystalline fatty substances which is less than 1% by weight.

This conversion to the molten state makes it possible to obtain, on theone hand, a phase containing the fatty substances and with a reducedcontent of minor fatty compounds and, on the other hand, a mixedwater-lipid phase containing the cyclodextrin/minor fatty compoundinclusion complexes.

These two phases separate from each other very well and it is thereforeeasy to collect the fatty phase free of the minor fatty compounds, whichmay then be used in various applications. The mixed phase, for its part,may be retreated to extract therefrom the minor fatty compounds whichmay be upgraded, especially in the pharmaceutical or cosmetics field.

When the substance to be treated is an animal and/or vegetable fat witha high lipid content such as for example butter which is concentrated orunconcentrated, tallow, lard, chicken fats, lanolins, hydrogenatedvegetable oils, palm or copra fats, margarines and the like, it isintroduced into a dough type kneading device provided with means forregulating the temperature.

Water is then optionally incorporated therein insofar as the substanceof biological origin to be treated does not contain it in a sufficientamount for the implementation of the process in conformity with theinvention. The amount of water required is preferably between 20 and 60%by weight relative to the substance of biological origin to be treated,expressed on a dry basis.

Cyclodextrin is then added, preferably in pulverulent form and insufficient amount for it to be partially in an undissolved form at thereaction temperature and to form inclusion complexes with the minorfatty compounds to be extracted.

The determination of the appropriate amount of cyclodextrin to be usedis a function, on the one hand, of the maximum solubility ofcyclodextrin at the temperature considered and, on the other hand, ofthe amount of minor fatty compounds to be extracted, and of theiraffinities for cyclodextrin. Generally, the amount of cyclodextrin is0.5% to 15% relative to the substance of biological origin to betreated, expressed on a dry basis, preferably from 1 to 10% and morepreferably from 3 to 8%.

The kneading of the paste formed by the mixing of these constituents iscarried out at a temperature which is maintained constant by regulatingmeans and chosen such that the fatty substance is in the congealed statewith a level of solid or crystalline fats above 5%, preferably above 10%and more preferably between 30 and 50% by weight. For concentratedbutter for example, the temperature is preferably chosen between about10° and 25° C.

After a variable time, generally between 10 and 120 min, the kneading isstopped and the paste is heated sufficiently so that the level of solidsin the fatty substance is less than 1% by weight. A water-lipid pelletcontaining the complexes and an oily liquid supernatant consisting ofthe fatty substance with a reduced minor fatty compound content arethereby formed.

As far as this preferred embodiment for the process according to theinvention is concerned, it should be noted that this process can beperfectly integrated within the framework of the manufacture of butterfrom cream. This manufacture essentially comprises the following stages:

biological or physical maturation (NIZO process) of the cream;

continuous or batch churning with emulsion reversal and separation ofbuttermilk;

optional kneading with biological maturation (NIZO process);

optional washing with water.

In conformity with the invention, cyclodextrin may be added afterseparation of the buttermilk, during the kneading. At the end of aplacing in contact which is carried out at the churning temperature(less than 15° C.), the separation of the inclusion complexes formed iscarried out by reverting to the molten state.

According to another embodiment of the process according to theinvention, the placing in contact of cyclodextrin with the substance tobe treated is carried out at a temperature chosen such that the fattysubstances constituting the said substance are in a noncongealed state.

In this embodiment of the process according to the invention, thesubstance to be treated, water and cyclodextrin, preferably introducedin this order, are mixed with stirring at a temperature chosen so thatthe fatty substances present are liquid (solids level less than 5% andpreferably less than 1%).

The temperatures corresponding to a solids level of less than 1% arepreferably less than 70° C. for most of the fatty substances. Thedetermination of the minimum temperature for the placing in contact iswholly within the scope of a person skilled in the art. Obviously, itdepends on the nature of the fatty substance considered,

The amount of water required is, preferably, between 45 and 80% byweight relative to the substance of biological origin to be treatedexpressed on a dry basis.

This embodiment is particularly suitable for the treatment of oilshaving a solids level of less than 1% at room temperature, such assunflower, soya bean, colza and maize type vegetable oils and the likeor such as fish oils.

The time of placing in contact ranges preferably between 10 and 120 min.

To separate the inclusion complexes formed, a centrifugation or adecantation of the mixture obtained is for example carried out after theplacing in contact, so as to recover a pellet containing the saidcomplexes, an aqueous intermediate phase and a supernatant lipid phasewith a reduced minor fatty compound content.

The above two embodiments of the process according to the inventionenable steroids and in particular sterols to be extracted with yields ashigh as about 90% in a single treatment. These yields may be improved byincreasing the number of treatments. These extraction efficiencies areobtained economically and easily in a relatively short time.

Another advantage of the process conforming to the invention is that itcan permit the selective extraction of a certain number of minor fattycompounds from a substance of biological origin, in a defined order, bycontrolling one or more essential process parameters, namely inparticular the amount of water present at the time of the complexingstage, the amount of cyclodextrin, the mixing or kneading temperature,the temperature for the change of phase, the pH of the medium.

Naturally, the invention is not limited to the embodiments describedabove but, on the contrary, it encompasses all the variants thereof.

The following examples will make it possible to understand more clearlythe process according to the present invention by demonstrating all theadvantages offered by it.

EXAMPLE I Extraction of Sterols, and More Particularly Cholesterol,which are Present in a Concentrated Butter in Conformity with thePreferred Embodiment of the Process According to the Invention

The concentrated butter used has a concentration of fatty substances ofthe order of 99.9%. The standard concentrated butter marketed by CORMANis chosen for example.

The cyclodextrin is a beta-cyclodextrin of the type marketed by theapplicant company under the registered trade mark KLEPTOSE and providedin the form of a white powder containing about 12% moisture.

1. Placing in contact

300 g of concentrated butter are introduced into the container of adough mixer of the type marketed by KUSTNER. The container of the mixeris provided with a double wall in which a temperature-regulating fluidmay be circulated. The mixer is started and the temperature ismaintained at a mean value of about 14° C. At this temperature, theconcentrated butter has a level of solid fats of about 28%.

150 g of water and 5.70 g of KLEPTOSE cyclodextrin are then addedsuccessively. Knowing that the cyclodextrin used contains 12% water, theconcentration of aqueous solution in the cyclodextrin is ##EQU2##

At the temperature for placing in contact (14° C.), the maximumsolubility of the beta-cyclodextrin is 1.20 g per 100 g of solution.This amounts to saying that in our example the level of cyclodextrinsaturation is 2.75.

The placing in contact then proceeds for 30 minutes.

2. Separation of the inclusion complexes

The pasty concentrated butter/water/cyclodextrin mixture removed fromthe mixer is heated to a temperature of the order of 37° C. (level ofsolid fats of the concentrated butter less than 1%). A change of phaseoccurs spontaneously and a pasty pellet is obtained containing inparticular the inclusion complexes and water. This pellet is covered byan oily phase formed by the decholesterolised concentrated butter. Morethan 94% by weight of the concentrated butter used can then berecovered.

The cholesterol is assayed by a colorimetric method after oxidation tocholestenone using cholesterol oxidase, according to the method of, andusing the reagents with the reference no. 139 050 from, BOEHRINGERMannheim.

The initial cholesterol content of the concentrated butter is 0.270% byweight.

The final content is 0.038%.

The extraction yield therefore amounts to 86%.

A second extraction, carried out under the same conditions, provides ayield of 74%, which gives an overall yield of 96%.

It can be observed that the efficiency of the process according to theinvention is quite remarkable both from the point of view of the amountand that of the duration.

The amount of water used is quite small and poses no problem ofhandling. The pasty pellet containing water which is used representsonly 173 g. This small and concentrated amount poses no problem withrespect to its retreatment.

EXAMPLE II Extractions of Sterols and More Particularly Cholesterol,which are Present in Concentrated Butter in Conformity with thePreferred Embodiment of the Invention

In this example, 9 tests for extracting cholesterols from concentratedbutter were carried out using the same equipment and by following thesame procedure as in Example I. Certain experimental parameters weresimply changed. The data and results of these tests are presented inTable I below:

                                      TABLE I                                     __________________________________________________________________________     Tests                                                                            ##STR1##                                                                             in °C.peratureMean tem-                                                      M.S.*                                                                             ##STR2##                                                                              minutesin contact in Duration of                                                        (3)Yield                                                                          saturationLevel                    __________________________________________________________________________                                              of                                  1   2060  15    1.20                                                                              515      30       65  17.80                               2   3090  15    1.20                                                                              515      30       85  12.00                               3   50150 15    1.20                                                                              515      30       84   7.25                               4  100300 15    1.20                                                                              515      30       75   3.64                               5   50150 16    1.20                                                                              721      30       93  10.10                               6   50150 16    1.20                                                                              515      15       75   7.25                               7   50150 26.5  1.80                                                                              515      30       65   4.83                               8   50150 16    1.20                                                                              39       30       59   4.37                               9   50150 16    1.20                                                                              39      120       55   4.37                               __________________________________________________________________________     *M.S. = maximum solubility at given temperature                               *CD = cyclodextrin                                                            (1) % relative to concentrated butter                                         (2) % relative to concentrated butter                                         (3) Final cholesterol/initial cholesterol extraction yield (in %)        

Comments on Table I

By comparing the results of tests 1 to 4 which differ only in the amountof water used, it can be observed that the cholesterol extraction yield,expressed in %, is maximum for water contents of between 30 and 50%relative to concentrated butter. The increase in the water contentbrings about a drop in the extraction yield.

By comparing the results of tests 3 and 5 for which only the KLEPTOSEcyclodextrin concentration is different (5 and 7% respectively relativeto concentrated butter), it can be observed that it is possible toincrease the extraction yield by 9% purely by increasing thecyclodextrin concentration by 2%.

In test 6, the duration of kneading was reduced to 15 minutes. The yieldobtained is 75%, which constitutes a very satisfactory result.

For test 7, the temperature of placing in contact was increased to 26.6°C. By reducing the solids level in the concentrated butter, thecholesterol extraction yield is reduced (65% against 84% for test 3).

By using 3% cyclodextrin relative to concentrated butter (tests 8 and9), it can be observed that beyond 30 min, prolonging the kneading doesnot enable the yields to be increased.

EXAMPLE III Extraction of Sterols, and More Particularly Cholesterol,which are Present in Butter in Conformity with the Preferred Embodimentof the Process According to the Invention

The butter used in this example is a commercial butter containing 82% offatty substances.

The cyclodextrin, the equipment as well as the procedure are the same asfor the preceding examples, the only difference being that no water isadded; it is the butter itself which provides the water required forcomplex formation.

The data and results of this example are given in Table II below:

                                      TABLE II                                    __________________________________________________________________________     ##STR3##   in °C.peratureMean tem-                                                      M.S.*                                                                             ##STR4##                                                                              in contactDuration of placing                                                           yield in %Extraction                                                                saturationLevel                 __________________________________________________________________________                                                 of                               Butter                                                                            20.525.5                                                                             12.5  1.10                                                                              57.5    45        80    22.73                            150 g                                                                         __________________________________________________________________________     *M.S. = maximum solubility at given temperature                               *CD = cyclodextrin                                                            (1) % relative to concentrated butter                                         (2) % relative to concentrated butter                                    

It is shown here that the process according to the invention is alsoeffective on a substance to be treated initially containing water in anamount conforming to the characteristics of the present invention.

EXAMPLE IV Extraction of Sterols, and More Particularly Cholesterol,which are Present in Tallow, in Conformity with the Preferred Embodimentof the Process According to the Invention

200 g of tallow containing 99% of fatty substances, of the type marketedby ROUSSELOT, are treated in the same manner as in the precedingexamples.

The initial cholesterol content of the tallow is 0.12% by weight.

Table III below shows the data and the results of the present example.

                                      TABLE III                                   __________________________________________________________________________     ##STR5##   in °C.peratureMean tem-                                                      M.S.*                                                                             ##STR6##                                                                              in contactDuration of placing                                                           yield in %Extraction                                                                  saturationLevel               __________________________________________________________________________                                                   of                              Tallow                                                                             50100                                                                               16    1.20                                                                              36      30        83                                                                                   4.36                                                        60        82                                      200 g                                                                              2550 16     1.20                                                                              36      30        86                                                                                   8.70                                                        60        84                                     __________________________________________________________________________     *M.S. = maximum solubility at given temperature                               *CD = cyclodextrin                                                            (1) % relative to concentrated butter                                         (2) % relative to concentrated butter                                    

Comments on Table III

It appears that an amount of water of 25% by weight relative to thetallow is sufficient to extract 86% of the initial cholesterol, and thiswith only 3% by weight of cyclodextrin relative to the tallow.

An increase in the amount of water and/or the duration of placing incontact does not enable the extraction yield to be improved.

In this example, the variations in the acid values were also measured.The acid value is defined as the number of mg of potassium hydroxiderequired to neutralise the acidity of 1 g of tallow. The acid value oftallow before extraction is 0.85.

Table IV below presents the results obtained.

                  TABLE IV                                                        ______________________________________                                        Water      Tem-   CD        Duration     % re-                                (% rela-   pera-  Kleptose  of plac-                                                                             Final duction                              tive to    ture   (% relative                                                                             ing in acid  in acid                              tallow)    °C.                                                                           to tallow)                                                                              contact                                                                              value value                                ______________________________________                                        Tallow                                                                              50       26     3       30     0.65  30                                 200 g 25       16     3       30     0.12  86                                 ______________________________________                                    

It is shown here that the decrease in acid value, and therefore of thefree fatty acid content, of the tallow is very pronounced with 25% ofwater.

EXAMPLE V Extraction of Sterols, and more Particularly Cholesterol,which are Present in a Fish Oil, in Conformity with the PreferredEmbodiment of the Process According to the Invention

The fish oil used is a salmon oil having a cholesterol content of 0.71%.In a first test, 300 g of this oil are mixed with 150 g of ice flakesand 30 g of KLEPTOSE cyclodextrin. The extraction treatment is thencarried out as described in Example 1. The temperature of placing incontact is 4° C.

In a second test, 120 g of this oil are premixed with 180 g ofhydrogenated copra oil for 20 minutes at 18° C. The mixture of fattysubstances obtained is then subjected to the extraction processconforming to that described in the preceding examples using 15 g ofKLEPTOSE cyclodextrin and 90 g of water.

The results and data for these two tests are collated in Table V.

                                      TABLE V                                     __________________________________________________________________________    Fish oils/                             Level                                  fatty sub-                     Tem-    of ex-                                                                             Level                             stances  % water/                                                                            % Kleptose                                                                          % Kleptose                                                                              perature                                                                              traction                                                                           of satura-                        treated  FS    CD/FS CD/fish oil                                                                         M.S.*                                                                             in °C.                                                                      Time                                                                             in % tion                              __________________________________________________________________________    Test 1                                                                            100% 50    10    10    0.90                                                                               4   30 27   19.10                             Test 2                                                                             40% 30     5    12.5  1.40                                                                              18   30 66   10.27                             __________________________________________________________________________     *M.S. = = maximum solubility at given temperature                        

By adjusting the level of solids by adding hydrogenated copra oil, theextraction yield of the cholesterol initially present in the salmon oilis improved.

EXAMPLE VI Extraction of Fat-Soluble Pigments, Flavouring Compounds andDegradation Compounds which are Present in Frying Fat and in a Fish Oil,in Conformity with the Preferred Embodiment of the Process According tothe Invention

A vegetable fat for frying which is marketed by LESIEUR under the brandname VEGETALINE is used to carry out 12 fryings spaced out over time:

2 fryings of breaded fish,

3 fryings of sliced potatoes,

3 fryings of whiting fillets,

1 frying of pork back fat,

3 fryings of meat croquettes.

This fatty substance is treated with and without KLEPTOSE β-cyclodextrinaccording to the procedure described in Example 1.

The odour and the colour of the products after treatment are evaluatedqualitatively. The results are given in Table VI below.

The level of degradation of the fatty substances is assessed by usingthe OXIFRIT-TEST® test marketed by MERCK.

A fish oil from which mucilages have been removed is treated in the samemanner at low temperature. The odour and the colour of this oil, treatedor untreated with β-cyclodextrin, are given in Table VI.

                                      TABLE VI                                    __________________________________________________________________________                                             Colour                                              % β-cyclo-           in the                               Nature   %/water/                                                                            dextrin/  Temp.                                                                             Time                                                                             Temp.                                                                             Odour                                                                              molten                                                                             Result                          (1)   Test                                                                             mg    mg    M.S.*                                                                             (2) (3)                                                                              (4) at 20° C.                                                                   state                                                                              (5)   L.S.*                     __________________________________________________________________________    Vegetable                                                                           1  50    0     --  16  30 60  Very Chest-                                                                             To be --                        F.S. after                          strong                                                                             nut- changed                         12 fryings                          frying                                                                             coloured                                                                           (dark                                                               odour     green)                                2  50    5     1.20                                                                              16  30 60  faint                                                                              Cream                                                                              Still 7.24                                                          odour                                                                              coloured                                                                           good                                                                          (emerald)                       Unrefined                                                                           3  30    0     --  3   30 60  Rancid                                                                             Dark --    --                        fish oil                            and  brown                                from which                          pungent                                   mucilages                           odour                                     have been                                                                           4  30    5     0.90                                                                              3   30 60  Odour of                                                                           Yellow                                                                             --    16.0                      removed                             fresh                                                                         fish                                      __________________________________________________________________________     *M.S. = maximum solubility at given temperature                               *L.S. = level of saturation                                                   *F.S. = fatty substance                                                       (1) Nature of the substance treated                                           (2) Treatment temperature in °C.                                       (3) Implementation time                                                       (4) Separation temperature in °C.                                      (5) Result of the OXIFRIT ® test                                     

It can be observed that the two fatty substances treated withβ-cyclodextrin in conformity with the invention exhibit betterorganoleptic properties.

As far as the frying fat is concerned, the treatment with β-cyclodextrinallowed removal of oxidised compounds (hydroperoxides, aldehydes,ketones, sterones and the like)

EXAMPLE VII Extraction of Sterols, and More Particularly Cholesterol,which are Present in Concentrated Butter, in Conformity with the SecondEmbodiment of the Process According to the Invention

In this example, the concentrated butter and cyclodextrin used areidentical to those of Examples I and II.

1. Procedure

300 g of concentrated butter containing 0.27% cholesterol are introducedinto a container equipped with stirring means and heated to atemperature of 50° C. 150 g of water and 15 g of KLEPTOSE cyclodextrinare then introduced therein. The placing in contact proceeds for 30 minwith stirring and with the temperature being maintained at 50° C. Thelevel of saturation is ##EQU3##

2. Separation of the complexes

The concentrated butter/KLEPTOSE cyclodextrin/water mixture is subjectedto a centrifugation at 3000 g for 5 minutes. The centrifugation enablesthe mixture to be separated into three phases:

a pasty pellet containing the cyclodextrin/steroids inclusion complexes;

an aqueous intermediate phase;

and a supernatant phase with a reduced sterol content.

After washing the supernatant phase with an equal amount of water, acholesterol extraction yield of 80% is determined.

It appears that the process conforming to the invention permits asubstantial reduction in the sterol content with a limited amount ofwater.

EXAMPLE VIII Extraction of Sterols, and More Particularly Cholesterol,which are Present in Concentrated Butter, in Conformity with the SecondEmbodiment of the Process According to the Invention

In this example, 6 tests for extracting sterols from concentrated butterare carried out in the same manner as in Example VII. Certainexperimental parameters were simply changed. The data and results ofthese tests are presented in Table VII below:

                  TABLE VII                                                       ______________________________________                                                     Kleptose                                                                      CD %                                                                    Temp. relative                                                              Water   M     S   to the       Time Yield Level                          Tests                                                                              (1)     (2)     AMF*   M.S.* (3)  (4)   (5)                              ______________________________________                                        1    50      50    50  1      4.64  30   26    0.38                                "       "     "   2      "     "    33    0.75                                "       "     "   5      "     "    80    1.87                                "       "     "   7      "     "    88    2.60                           2    60      45    50  7      4.10  30   83    2.47                                "       55    "   "      6.05  "    80    1.67                                "       60    "   "      7.49  "    83    1.35                                "       65    "   "      10.8  "    71    0.94                                "       70    "   "      12.03 "    56    0.84                           3    60      60    60  7      7.49  30   83    1.35                                "       "     75  "      "     "    73                                        "       "     80  "      "     "    60    "                                   "       "     90  "      "     "    37    "                              4    20      50    50  5      4.64  30   44    4.60                                30      "     "   "      "     "    58    3.10                                40      "     "   "      "     "    63    2.33                                50      "     "   "      "     "    80    1.87                                85      "     "   "      "     "    70    1.11                           5    60      50    50  5      4.64   2   38    1.56                                "       "     "   "      "      12  78    "                                   "       "     "   "      "      30  80    "                                   "       "     "   "      "     105  81    "                                   "       "     "   "      "     180  80    "                                   "       "     "   "      "     285  79    "                              ______________________________________                                         *AMF = anhydrous milk fat                                                     *M.S. = maximum solubility at given temperature                               (1) in % relative to the AMF                                                  (2) temperature in °C.                                                 M = placing in contact - S = separation                                       (3) time of placing in contact in minutes                                     (4) extraction yield                                                          final cholesterol                                                             ##STR7##                                                                      initial cholesterol                                                           (5) level of saturation                                                  

Comments on the Results

In test 1, cyclodextrin concentrations of 1 to 7% by weight relative tothe concentrated butter resulted in extraction yields of between 26 and88%. The extraction yields are improved by increasing the level ofsaturation.

Test 2 shows that the higher the increase in the temperature of placingin contact and the greater the decrease in the saturation level, thegreater the decrease of the corresponding extraction yields.

Test 3 shows that the same applies for the separation temperature. Itseems preferable not to exceed 60° C.

The results of test 4 show that an amount of water equal to 50% enablesa very satisfactory extraction yield of 80%, higher than that obtainedwith 100% water, to be achieved.

The extractions carried out with increasing periods of placing incontact, in test 5, show that from a time of the order of 12 minutesupwards, a plateau is practically reached which is equivalent to anextraction yield of about 80%.

EXAMPLE IX Treatment of a Fatty Medium with Cyclodextrin

An amount of 50 kg of maize oil, whose phospholipids have been removedbeforehand (removal of mucilages) and which has an acid value of 1.5(expressed in % oleic acid per 100 g), is treated with 1.2 kg ofβ-cyclodextrin (marketed by the applicant company under the brand nameKLEPTOSE) so as to extract the free fatty acids and the degradationcompounds which are present in this oil.

The β-cyclodextrin, which is provided in powdered form, is dispersed inthe oil to be treated by stirring. 20 kg of water are slowly added so asto form a crude emulsion by stirring and at room temperature (20° C.,M.S.=1.48). The level of saturation is 3.54.

The whole mixture is then homogenised by stirring at 13,500revolutions/minute for 1 minute using a UNIMIX type apparatus. A fineemulsion of the oil-in-water type with a texture similar to that ofmayonnaise is thus obtained.

This emulsion is heated to 90° C. before being centrifuged at 6000revolutions/minute for 5 minutes. Three phases are thus formed:

an oily phase which, after washing with water and drying, has an acidvalue of 0.6,

an aqueous phase,

a centrifugation pellet which is white in colour.

EXAMPLE X Treatment of a Deodorisation Condensate using Cyclodextrin

1000 g of deodorisation condensate of a sunflower oil are treated withβ-cyclodextrin (marketed under the registered trade mark KLEPTOSE by theapplicant company) so as to extract the phytosterols which it contains.The composition of this coproduct which is derived from the refining ofa sunflower oil is given in Table III below.

A double extraction is carried out on this condensate by proceeding asfollows:

160 g of KLEPTOSE β-cyclodextrin and 160 g of water at 20° C.(M.S.=1.48) are added to the condensate. The level of saturation is 53.The mixture is homogenised for 5 minutes using an ULTRATURAX typeequipment so as to produce a viscous and homogeneous emulsion. Thisemulsion is then diluted with 3000 g of water before being centrifugedat 3000 revolutions/minute and at room temperature. A firstcentrifugation pellet, a fatty phase and an intermediate aqueous phaseare thus separated. The latter is removed. The other two phases areretained.

160 g of KLEPTOSE β-cyclodextrin and 160 g of water at 20° C. are addedto the supernatant fatty phase obtained as described above so as toachieve a second extraction. The same procedure is followed in order toobtain a second centrifugation pellet and a fatty phase which has beensubjected to a double extraction. This fatty phase is dried in an ovenat 40° C. for 48 hours.

Its composition and that of the starting condensate are given in TableVIII below.

                  TABLE VIII                                                      ______________________________________                                                    Composition of                                                                            Composition of                                                    the deodorisation                                                                         the supernatant                                                   condensate  fatty phase                                                       used (%)    (%)                                                   ______________________________________                                        Total fatty substances                                                                      86.0          94.0                                              including                                                                     free fatty acids                                                                            82.0          82.5                                              and triglycerides                                                             phytosterols  13.4          12.8                                              (β sitosterol +                                                          stigmasterol)                                                                 tocopherols    4.6           4.7                                              (α+ β)                                                             β-cyclodextrin                                                                         --                                                              Others        14.0           6.0                                              (water, glycerine,                                                            pigments)                                                                     ______________________________________                                    

The β-cyclodextrin, through selective inclusion, permitted extraction ofthe phytosterols. The fatty phase treated is enriched with tocopherols,free fatty acids and triglycerides.

EXAMPLE XI Production of an Enriched Aromatic Isolate using Cyclodextrin

A peppermint essential oil is treated with β-cyclodextrin (marketed bythe applicant company under the brand name KLEPTOSE) so as to obtain amenthol-rich aromatic isolate.

For this, 110 g of KLEPTOSE β-cyclodextrin are mixed with 50 g ofdistilled water at room temperature (M.S.=1.48). The level of saturationis 103.5. A viscous white paste is thus obtained to which 200 g ofessential oil are slowly added with mechanical stirring.

The mixture is then homogenised at 13,500 revolutions/minute using aUNIMIX type apparatus. A paste, in suspension in the oily medium, isthus obtained.

This paste is separated from the excess oil by filtration under vacuumusing a Buchner type apparatus. A white aromatic powder is finallyobtained. The said powder contains, on a dry basis, 12.6% aromaticisolate.

We claim:
 1. A process for extracting minor fatty compounds selectedfrom the group consisting of steroids, fatty alcohols, fat solublevitamins, pigments, hydrocarbons and flavouring compounds from asubstance of biological origin containing fatty substances, comprisingthe steps of:selecting a substance of biological origin containing saidminor fatty compounds and fatty substances; selecting a cyclodextrin inpulverulent form capable of forming inclusion complexes with said minorfatty compounds, from the group consisting of substituted andunsubstituted alpha, beta and gamma cyclodextrin; providing an amount ofsaid cyclodextrin of from 0.5 to 15% relative to the dry matter of thesubstance of biological origin to be treated; contacting the amount ofcyclodextrin with the substance of biological origin in an aqueousreaction medium at a temperature about 10° C. to 25° C. and maintainingthe water content of the reaction medium so as to obtain a saturationlevel of cyclodextrin in water equal to or greater than 1.67 at thereaction temperature and so that the cyclodextrin is in a partiallyundissolved form; mixing during 10 to 120 minutes the cyclodextrin withthe substance of biological origin so as to obtain a pasty mixture andto allow the formation of the said inclusion complexes; separating, thesaid inclusion complexes formed during the contacting step from the saidsubstance of biological origin by centrifugation, by decantation, or byheating the mixture obtained to a temperature at which not more than 1%of the fatty substances present in the substance of biological originare in a solid or crystalline state; and recovering the substance ofbiological origin with a reduced content of minor fatty compounds. 2.The process according to claim 1, wherein the saturation level ofcyclodextrin in water is between 1.87 and
 750. 3. The process accordingto claim 1, wherein the saturation level of cyclodextrin in water isbetween 2 and
 750. 4. The process according to claim 1, wherein theamount of cyclodextrin is 0.5 to 15% relative to the dry matter of thesubstance of biological origin to be treated.
 5. The process accordingto claim 1, wherein the amount of cyclodextrin is 1 to 10% relative tothe dry matter of the substance of biological origin to be treated. 6.The process according to claim 1, wherein the amount of cyclodextrin is3 to 8% relative to the dry matter of the substance of biological originto be treated.
 7. The process according to claim 1, wherein the amountof water present in the reaction medium is between 20% and 80% parweight relative to the dry matter of the substance of biological originto be treated.
 8. The process according to claim 1, wherein thecyclodextrin is of the α type.
 9. The process according to claim 1,wherein the cyclodextrin is of the β type.
 10. The process according toclaim 1, wherein the temperature during the contacting step is thetemperature at which at least 5% by weight of the fatty substancespresent in the substance of biological origin are in a solid orcrystalline state.
 11. The process according to claim 1, wherein thetemperature during the contacting step is the temperature at which atleast 10% by weight of the fatty susbstances present in the substance ofbiological origin are in a solid or crystalline state.
 12. The processaccording to claim 1, wherein the temperature during the contacting stepis the temperature at which from 30% to 50% by weight of the fattysubstances present in the substance of biological origin are in a solidor crystalline state.
 13. The process according to claim 1, wherein themixture of substance of biological origin with water and withcyclodextrin is subjected, during the contacting step, to a kneadingoperation during which said complexes of cyclodextrin with minor fattycompounds are formed.
 14. The process according to claim 1, wherein theseparation of the complexes of cyclodextrin with minor fatty compoundsis carried out by heating the reaction medium to a temperature at whichat most 1% of the fatty substances present in the substance ofbiological origin are in a solid or crystalline state and by separating,on the one hand, the phase containing the fatty substances with areduced content of minor fatty compounds and, on the other hand, themixed water-lipid phase containing the complexes between thecyclodextrin and the minor fatty compounds.
 15. The process according toclaim 1, wherein the temperature during the separation step is less thanor equal to 60° C.
 16. The process according to claim 1, wherein thesubstance of biological origin is an animal fat or a vegetable fathaving a total phospholipid level less than 10% by weight relative tothe total lipids present in said substance of biological origin.
 17. Theprocess according to claim 1, wherein the substance of biological originis selected in the group consisting of concentrated butter, butter,tallow, fish oils, frying fats, lanolins, deodorisation condensates andessential oils.
 18. The process according to claim 17, wherein thesubstance of biological origin is concentrated butter.
 19. The processaccording to claim 1 wherein the separation of the complexes ofcyclodextrin with minor fatty compounds is carried out by centrifugationor by decantation so as to recover a pellet containing the saidcomplexes, an aqueous intermediate phase and a supernatant lipid phasewith a reduced minor fatty compounds content.
 20. A method according toclaim 1 further comprising the step of diluting said pasty mixture withwater prior to separating the infusion complexes from the substances ofbiological origin.